TaqMan Protein Assays Probe Development

QUICK REFERENCE CARD TaqMan Protein Assays Probe Development Note: For safety and biohazard guidelines, refer to the Safety section in the TaqMan Protein Assays Probe Development Protocol (Part no. 4449282). For every chemical, read the Safety Data Sheet (SDS) and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Workflow for the Forced Proximity Probe Test Antibody dilution & Prox-Oligo mix preparation Dilute biotinylated to 200 nm For example: 47 µl Antibody Dilution uffer 3 µl 0.mg/mL iotinylated Prepare Prox-Oligo mix 3 Prox-Oligo Prox-Oligo Probe & Negative Control preparation Prepare Forced Proximity Probe 2 µl 200 nm Prox-Oligo mix 2 µl 200 nm iotinylated Prepare Negative Control 2 µl 200 nm Prox-Oligo mix 2 µl Antibody Dilution uffer 60 min Add 396 µl Assay Probe Dilution uffer 30 min Ligation reaction Dilute ligase 2 µl DNA Ligase 198 µl Ligase Dilution uffer Prepare ligation solution 0 µl Ligation Reaction uffer 908 µl Water 2 µl Diluted DNA Ligase Plate ligation reactions 4 µl/well Forced Proximity Probe or Neg. Control 96 µl/well ligation solution 37 C, 10 min Protease reaction Stop ligation 1 µl Protease 99 µl PS Add 2 µl/well 37 C, 10 min 9 C, min PCR Prepare PCR mix 100 µl TaqMan Protein Assays Fast Master Mix 10 µl Universal PCR Assay Plate PCRs 11 µl/well PCR mix 9 µl/well proteasetreated ligation products Perform real-time PCR Analysis Analyze with automatic baseline and a threshold cycle of 0.2 Export the C T data Passing criteria ΔC T 8. Workflow for preparing Assay Probe A and Assay Probe Assay Probe A preparation Prepare Assay Probe A 3 Prox-Oligo iotinylated Assay Probe preparation Prepare Assay Probe Prox-Oligo iotinylated 60 min 60 min Add 90 µl Assay Probe Storage uffer 20 min Add 90 µl Assay Probe Storage uffer 20 min

Select and prepare biotinylated antibodies Select and prepare biontinylated antibodies according to the TaqMan Protein Assays Probe Development Protocol. Perform the Forced Proximity Probe Test 1 Dilute the biotinylated to 200 nm (30 µg/ml) a. If you are using frozen biotinylated : Remove an aliquot from storage and immediately place it on ice to thaw. After the has thawed, gently mix (but do not vortex). Spin the tube briefly (~ seconds) to bring the liquid to the tube bottom. b. Working on ice, combine the components listed below. riefly centrifuge to spin the liquid to the tube bottom, then place on ice. EXAMPLE For stock solution at 0. mg/ml (3.3 µm) Antibody Dilution uffer 47 µl iotinylated stock solution 3 µl Total volume of 200 nm biotinylated 0 µl After diluting the biotinylated : Proceed to Prepare the Forced Proximity Probe and the negative control below. You need 2 µl of the 200 nm biotinylated to prepare the Forced Proximity Probe. AND Store the rest of the 200 nm biotinylated at 4 C for up to 48 hours. After the biotinylated has passed the Forced Proximity Probe Test, you can use the 200 nm biotinylated to prepare the Assay Probes (page 6). 2 Prox-Oligo A Prox-Oligo Prepare the Forced Proximity Probe and the negative control a. Prepare the Prox-Oligo mix: Working on ice, combine the components listed below. Mix gently, then briefly centrifuge to spin the liquid to the tube bottom. Place on ice. 200 nm 3 Prox-Oligo µl 200 nm Prox-Oligo µl Total volume of 200 nm Prox-Oligo mix 10 µl b. Label two tubes: Forced Proximity Probe and Negative Control. c. Working on ice, combine the components listed below in the appropriate tube. Mix gently, then briefly centrifuge to spin the liquid to the tube bottoms. Forced Proximity Probe Negative Control 200 nm Prox-Oligo mix 2 µl 2 µl 200 nm iotinylated 2 µl Antibody Dilution uffer 2 µl Total volume 4 µl 4 µl d. the tubes at room temperature for 60 minutes. 2

3 Perform the ligation reaction e. Add 396 µl of Assay Probe Dilution uffer to each tube. f. the tubes at room temperature for 30 minutes, then place both tubes on ice. Proceed to Perform the ligation reaction below. IMPORTANT! For step 3 (performing the ligation reaction) through step 6 on page (preparing the PCR plate): Keep all reagents on ice when not in use. Do not allow the tubes to warm to room temperature. Keep the reaction plates on ice during reagent transfers. a. Thaw or place the following components on ice: 00 DNA Ligase, 1 Ligase Dilution uffer, 20 Ligation Reaction uffer, 1 PS (ph 7.4), 100 Protease. b. Dilute the DNA Ligase: Combine the components listed below. Mix gently, then place on ice. IMPORTANT! Prepare fresh diluted ligase for each experiment. DNA Ligase, 00 2 µl Ligase Dilution uffer, 1 198 µl Total volume of diluted DNA Ligase 200 µl c. Prepare the ligation solution: Combine the components listed below. Invert the tube to mix, then place on ice. Ligation Reaction uffer, 20 0 µl Nuclease-free water 908 µl Diluted DNA Ligase 2 µl Total volume of ligation solution 960 µl d. Place a 96-well reaction plate on ice. To 8 wells of the plate, add the components as follows: Volume per well (µl) 1 2 3 e. Add 96 µl of the ligation solution to each of the 8 reaction wells. Pipet up and down once to mix. f. Seal the ligation reaction plate with a MicroAmp Clear Adhesive Film, then briefly centrifuge the plate. 4 6 7 Forced Proximity Probe 4 4 4 4 NA Negative Control NA 4 4 4 4 Total volume per well (µl) 4 4 4 4 4 4 4 4 8 3

g. the sealed reaction plate using the thermal-cycling conditions below. IMPORTANT! To prevent condensation and evaporation, you must use a compression pad and a heated cover when using the thermal cycler. Failure to do so will result in highly variable data. 4 Perform the protease reaction Step Cycle no. Temperature ( C) Time Reaction volume Ligation 1 37 C 10 minutes Default Cooling 1 4 C 10 minutes Default You can omit the protease step if you proceed immediately to the real-time PCR steps, beginning with Prepare the PCR mix on page. Otherwise, continue with the protease reaction below. a. Dilute the protease: 1. riefly vortex the protease to mix the solution. 2. Combine the components listed below. Mix gently, then briefly centrifuge to spin the liquid to the tube bottom. Place on ice. Protease, 100 1 µl 1 PS, ph 7.4 99 µl Total volume of diluted protease solution 100 µl b. Remove the ligation reaction plate from the thermal cycler, remove the MicroAmp Clear Adhesive Film, then place the plate on ice. c. Add 2 µl of the diluted protease to each of the 8 reaction wells of the ligation reaction plate. Note: No mixing is required. The protease will diffuse throughout the samples during the 10-minute incubation. d. Reseal the reaction plate with a new adhesive film. e. the sealed reaction plate using the thermal-cycling conditions below. IMPORTANT! To prevent condensation and evaporation, you must use a compression pad and a heated cover when using the thermal cycler. Failure to do so will result in highly variable data. Step Cycle number Temperature ( C) Time Reaction volume Terminate ligation 1 37 C 10 minutes Default Inactivate protease 1 9 C minutes Default HOLD 1 4 C Hold Default f. Remove the reaction plate from the thermal cycler and place it on ice. Proceed to Prepare the PCR mix on page OR store the protease-treated ligation products at 4 C for up to 3 days, or at 20 C for up to 2 weeks. 4

Prepare the PCR mix a. Thaw the Universal PCR Assay on ice. b. Combine the components listed below. Mix gently, then briefly centrifuge to spin the liquid to the tube bottom. Place on ice. Fast Master Mix, 2X 100 µl Universal PCR Assay, 20 10 µl Total volume of PCR mix 110 µl 6 Prepare the PCR plate a. Place a PCR plate on ice, then add 11 µl of the PCR mix to each of 8 wells of the PCR plate. b. Remove the adhesive film from the protease reaction plate, then place the plate on ice. c. Transfer 9 μl of the protease-treated ligation product from each of the 8 reaction wells of the protease reaction plate to each of the 8 reaction wells of the PCR plate. When transferring, pipet up and down once to mix. d. Seal the PCR plate with a MicroAmp Optical Adhesive Film, then briefly centrifuge the plate. For the 7900HT/7900HT Fast system with a 96- lock Module and automation accessory, place a MicroAmp Snap-On Optical Film Compression Pad on top of the plate. IMPORTANT! Proceed immediately to Run the PCR plate below. 7 Run the PCR plate In your real-time PCR system software, create a plate document/experiment for the run, using the setup information below. Load the PCR plate into your real-time PCR instrument, then start the run. System StepOnePlus 700 Fast 7900HT Fast 7900HT Software StepOne Software v1.0 or later SDS Software v1.4 or later 700 Software v2.0 or later SDS Software v2.1 or later Template cdna cdna Run type Fast Standard Reaction plate Fast 96-well Fast 96-well, Standard 96-well, or 384-well SDS Software v2.0 or later Standard 96-well or 384- well Sample volume 20 µl 20 µl Detectors/targets Reporter: FAM dye Quencher: Non-fluorescent Reporter: FAM dye Quencher: Non-fluorescent Ramp speed/mode Fast Fast or Standard Standard Experiment type Select an experiment type that will generate C T values, such as Absolute Quantitation or Standard Curve. Tasks and You do not need to assign Tasks or quantities. quantities Analysis settings Threshold cycle (C T ): 0.2; aseline: Automatic

8 Analyze the data Method Procedure Using the real-time PCR system software Using a spreadsheet program 1. View the amplification plots for the entire reaction plate. 2. Analyze the plate run using a threshold cycle (C T ) setting of 0.2 and automatic baseline. 1. Export the results from the instrument software to a spreadsheet program. 2. Calculate the average C T values for each biotinylated and negative control. 3. Calculate the ΔC T values for each biotinylated : AvgC T (negative control) AvgC T (Forced Proximity Probe) Expected Results ΔC T value Result Comment 8. Pass The biotinylated is suitable for use in TaqMan Protein Assays experiments. Prepare Assay Probe A or Assay Probe IMPORTANT! If you assemble Assay Probes A and at the same time, be extremely careful not to cross-contaminate the Prox-Oligos. Change the pipette tips between each addition. 1. riefly centrifuge the 200 nm biotinylated to spin the liquid to the tube bottom, then place the tube on ice. 2. riefly centrifuge the appropriate Prox-Oligo to spin the liquid to the tube bottom, then place the tube on ice. Use the: 200 nm 3 Prox-Oligo for Assay Probe A 200 nm Prox-Oligo for Assay Probe 3. Working on ice, combine the components listed below. For Assay Probe A For Assay Probe 200 nm iotinylated Prox-Oligo A 200 nm 3 Prox-Oligo NA Prox-Oligo 200 nm Prox-Oligo NA Total volume 10 Assay 10 probe A Assay probe 4. Mix gently, then briefly centrifuge to spin the liquid to the tube bottom.. the tube at room temperature for 60 minutes. 6. Allow the Assay Probe Storage uffer to come to room temperature. 7. Add 90 µl of Assay Probe Storage uffer to the tube, then mix gently. The total volume should be 100 µl. 8. riefly centrifuge to spin the liquid to the tube bottom. 9. the tube at room temperature for 20 minutes. 10. Store the Assay Probe at 20 C for up to 6 months. 6

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